Ab2873

The following primary antibodies were used:
Anti-CALD1 (mouse IgG1, MS-1251-P0, NeoMarkers, Fremont, CA, USA, 1:500), anti-COCH (rabbit IgG, HPA050122, Atlas Antibodies, Bromma, Sweden, 1:200), anti-CCN2 (goat IgG, sc14939, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:200), anti-ATP1B1 (mouse IgG2a, ab2873, Abcam, Cambridge, UK, 1:1000), anti-ATP1A1 (rabbit IgG, ab76020, Abcam, Cambridge, UK, 1:1000), anti-SLC12A2 (goat IgG, sc-21545, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:1000), anti-KCNJ16 (rabbit IgG, APC123, Alomone labs, Jerusalem, Israel, 1:1000), anti-CA2 (rabbit IgG, sc-25596, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:500), anti-GLUT1 (rabbit IgG, ab115730, Abcam, Cambridge, UK, 1:500), anti-COL2A1 (mouse IgG2b, sc-52658, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:100).
The following secondary antibodies were used: donkey anti-mouse IgG, Alexa Fluor Plus 488 (A32766, Invitrogen, Waltham, MA, USA, 1:500); donkey anti-rabbit IgG, Alexa Fluor Plus 555 (A32794, Invitrogen, Waltham, MA, USA, 1:500); and donkey anti-goat Alexa Fluor Plus 647 (A32849, Invitrogen, Waltham, MA, USA, 1:500).

Cell surface protein was isolated according to the manual of Pierce Cell Surface Protein Isolation Kit from Thermo Scientific, Bonn, Germany (Cat# 89881). Briefly, four 75-cm² T75 flasks of HEK293-T cells ((1) without DNA, (2) GluA1/pIRES2/EGFP, (3) GluA1/pIRES2/EGFP + PIP5K2A/pcDNA3, or (4) GluA1/pIRES2/EGFP + PIP5K2A(N251S)/pcDNA3) were transfected with or without the corresponding DNA using the calcium phosphate transfection method. Confluent cells (90–95 %) were incubated with Sulfo-NHS-SS-Biotin for 30 min at 4 °C. Cells were scraped and transferred to a single 50-ml tube after adding a quenching solution to each flask. Cells were centrifuged (4 °C, 500×g, 5 min) and transferred into a lysis solution. After sonication, cells were incubated for 30 min on ice, vortexing every 5 min. Cell lysate was centrifuged (4 °C, 10,000×g, 2 min); clarified supernatant was transferred to a prepared NeutrAvidin agarose column and incubated for 60 min at room temperature. After washing of the column, it was incubated with SDS-PAGE sample buffer containing DTT for 60 min at room temperature. Elution of the purified cell surface proteins was performed by centrifugation (4 °C, 1,000×g, 2 min). Samples were analyzed by Western blot. Antibodies were obtained from Abcam, Cambridge, UK (GluA1, #ab31232, β-Na⁺/K⁺-ATPase, #ab2873).